Biomedical subjects

F Widmer

Publications and source records attributed to F Widmer.

At least 19 recordsLinked to original sources

PubMed2006

Changes in lead availability affect bacterial community structure but not basal respiration in a microcosm study with forest soils.

This study investigates the effects of Pb during time on the bacterial communities of forest soils using water-extractable Pb concentrations in the soil solution as predictors of Pb bioavailability. In a microcosm experiment we applied increasing concentrations of Pb(NO(3))(2) solutions (0.5, 2, 8, 32 mM) to 5 forest soils of pH<5 and to a calcareous soil of pH>6.5. Sampling of the microcosms was performed after 3, 30 and 90 days of incubation. Community analysis included basal respiration rates and changes in the structure of the bacterial communities through T-RFLP fingerprinting. We also investigated functional stability in terms of resistance, expressed as the effects on basal respiration after 3 days of incubation, and of resilience, expressed as the recovery of bacterial community structure and of respiration rates after 90 days of incubation. Water-extractable Pb increased with time in most of the soils, in parallel with an increase of water-extractable dissolved organic carbon (DOC). The increased concentrations slightly affected bacterial community structure, although OTU (operational taxonomic unit) richness was not significantly reduced with Pb concentrations in any of the soils. The highest Pb treatment (32 mM) caused significant effects on basal respiration in some of the acidic soils, but no clear trend was observed in relation to increased Pb bioavailability with time. Resistance to Pb additions was evident in five of the six soils, but only two showed resilience after 90 days. This is the first study showing the effects of time on Pb bioavailability in soils and on the resulting reactions of the soil microbial communities.

DNA, Bacterial↗

PubMed2006

Thyrotoxicosis-induced prinzmetal variant angina.

We describe a postmenopausal women with new onset of variant angina caused by thyrotoxicosis due to Graves' disease. During exercise bicycle ergometry at 50 Watts, the patient developed typical angina with ST segment elevation in the precordial leads. A coronary angiogram revealed normal coronary arteries. Graves' disease with overt hyperthyroidism was diagnosed. After achieving an euthyroid state with administration of propylthiouracil, the symptoms resolved completely and the patient had a normal exercise capacity without electrocardiographic changes. Thus, we conclude that in patients with thyrotoxicosis, variant angina and normal coronary arteries, restoration of normal thyroid function may be curative.

Angina Pectoris, Variant↗

PubMed2005

Optimization of bulked AFLP analysis and its application for exploring diversity of natural and cultivated populations of red clover.

Landraces and wild populations of red clover (Trifolium pratense L.) may represent a significant yet poorly characterized genetic resource of temperate grasslands. A bulking strategy with amplified fragment length polymorphism (AFLP) markers was optimized to characterize 120 red clover populations in 6 different groups: Swiss wild clover populations, Mattenklee landraces, Mattenklee cultivars, field clover cultivars, Dutch wild clover populations, and Dutch landraces. Analysis of 2 bulked samples/population consisting of 20 plants each with12 AFLP primer combinations was found optimal for determining genetic diversity and relationships within and among red clover populations and groups. Swiss wild clover populations were clearly separated from all other red clover groups and variability within and among populations was shown to be particularly high in wild clover populations and Mattenklee landraces, emphasising their value as genetic resources for improvement of red clover cultivars, as well as for conservation and restoration of biodiversity. This study shows that the ancestry of red clover landraces is primarily found in introduced cultivars rather than in natural wild clover populations. In addition, the methodological considerations presented here may help improve diversity analyses using bulked samples.

Genetic Markers↗

PubMed2003

Swiss Mattenklee landraces, a distinct and diverse genetic resource of red clover (Trifolium pratense L.).

Genetic variability within and among 19 landraces and cultivars of red clover ( Trifolium pratense L.) was investigated by means of amplified fragment length polymorphism (AFLP) analysis in order to assess the potential value of Swiss Mattenklee landraces as genetic resources for plant breeding and the preservation of biodiversity. Populations were classified into three groups according to their origin and agronomic features: Mattenklee landraces (8), Mattenklee cultivars (8) and field clover cultivars (3). Analysis of molecular variance based on 276 polymorphic AFLP markers revealed 80% of total variability to be due to variability within populations while 12% were attributed to variability among groups. Stepwise discriminant analysis identified a subset of 126 AFLP markers which best separated individual plants into the three respective groups. Genetic distances between populations were considerably larger among groups than among populations within the same group, providing further evidence for the genetic distinction between Mattenklee landraces, Mattenklee cultivars and field clover cultivars. AFLP markers identified two landrace clusters, containing three and four populations respectively, which, together with one additional landrace, may sufficiently represent the genetic variability of all eight landraces investigated. The results of this study strongly suggest that Swiss Mattenklee landraces form a genetically distinct group of red clover. The data obtained provide criteria on how to efficiently manage, preserve and exploit Mattenklee germplasm.

Analysis of Variance↗

PubMed2001

A strategy for optimizing quality and quantity of DNA extracted from soil.

The efficiency of a bead beating method was studied in detail with regard to a variety of factors including beating time and speed, volume and temperature of the buffer, as well as amount and type of beads employed. The results presented here reveal that all of these parameters have a significant effect on yield and quality of DNA extracted from soils. Precise adjustment of extraction conditions allows for significantly higher yields of high quality DNA from soils than previously reported. We further evaluated the effect of the extraction conditions on the apparent soil microbial community structures, as observed by polymerase chain reaction (PCR) and RFLP. Differences in the fingerprints of DNA extracted under different conditions suggest that results could be biased when using gentle extraction procedures. Based on multiple subsequent extractions using very harsh extraction conditions, we propose a protocol for the quantification of the total DNA content in soils. Extractions from six soils of different texture and chemical characteristics with selected bead beating protocols revealed that the quality (fragment size and purity) of the extracted DNA was generally very good, but also depended on the soil characteristics. While a single, general protocol for optimal DNA recovery from all soils cannot be given, this study provides detailed guidelines on how to optimize the general method to obtain optimal DNA from individual soils.

Buffers↗

PubMed2000

Temporal and Spatial Distribution of the nifH Gene of N(2) Fixing Bacteria in Forests and Clearcuts in Western Oregon.

Decomposition of plant litter is a primary mechanism of nutrient recycling and redistribution in most terrestrial ecosystems. Previously we demonstrated by a nested PCR protocol that 20 distinctive nifH (the gene encoding nitrogenase reductase) HaeIII restriction fragment length polymorphism (RFLP) patterns were derived from bulk DNA associated with samples of plant litter and soil collected at one Douglas Fir (DF) forest [33]. Five of the nifH DNA patterns (II-VI) were dominant types in DF litter with characteristic fragments of 237-303 bp length, whereas samples from soil contained primarily seven other patterns 131-188 bp length (IX-XV). Here we report that the 237-303 bp fragments characteristic for forest litter could generally not be detected in plant litter or soil samples collected in clearcuts that adjoin the forest sites. The same fragments (237-303 bp) were also found in the litter at this DF forest site over 16 months and were consistently found in litter at 12 other DF forest or recent (<2 yrs) clearcut sites. However, trace to none of these fragments were detected in 6 clearcut (5-10 yrs) or different forest types (oak, alder) collected over a 200 km east-west direction in western Oregon, USA. Data suggest that the logging practice in DF forests that creates a clearcut removes a unique gene pool of nitrogen-fixing microorganisms. These organisms could potentially contribute more to nitrogen fixation in forest litter than litter from natural or invasive plants that grow in clearcuts [26]. </hea

Journal Article↗

PubMed2000

Substrate preference profiles of proteases released by allergenic pollens.

BACKGROUND: Pollens are important triggers for allergic asthma and seasonal rhinitis. We have recently reported that proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of epithelial integrity by proteases released following deposition of pollens on mucosal surfaces could promote sensitization and induce inflammation. OBJECTIVE: To compare protease activities released by allergenic pollens of various genera. METHODS: We used a rapid microassay which quantifies cleavage of dipeptide ester substrates to characterize the substrate preference profiles of serine proteases in diffusates of the pollens of perennial ryegrass (Lolium perenne), Kentucky blue grass (Poa pratensis), Bermuda grass (Cynodon dactylon), Western ragweed (Ambrosia spp.), white birch (Betula spp.) and Sydney golden wattle (Acacia longifolia). RESULTS: Comparison of the profiles revealed notable differences as well as similarities between serine protease activities released by these pollens. Diffusates of Kentucky blue grass pollen exhibited very high substrate preference for arginine and lysine. For other pollens, cleavage of the cysteine substrate was usually the most rapid and was associated with marked preference for leucine and methionine. There was considerable variation between these pollens in the rates of cleavage of the histidine substrate. In addition, we observed high rates of cleavage of arginine and lysine substrates by Acacia pollen diffusate. CONCLUSION: At least two dominant patterns of substrate preference are identifiable in the mixtures of proteases released by hydrated pollens. Purification of the proteases responsible for these patterns of activity will facilitate investigation of their role in airway epithelial injury and allergic disease.

Allergens↗

PubMed1999

Fingerprinting of mixed bacterial strains and BIOLOG gram-negative (GN) substrate communities by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR).

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities.

DNA Fingerprinting↗

PubMed1999

Comparison of Parental and Transgenic Alfalfa Rhizosphere Bacterial Communities Using Biolog GN Metabolic Fingerprinting and Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR).

> Abstract Rhizosphere bacterial communities of parental and two transgenic alfalfa (Medicago sativa L.) of isogenic background were compared based on metabolic fingerprinting using Biolog GN microplates and DNA fingerprinting of bacterial communities present in Biolog GN substrate wells by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). The two transgenic alfalfa expressed either bacterial (Bacillus licheniformis) genes for alpha-amylase or fungal (Phanerochaete chrysosporium) genes for Mn-dependent lignin peroxidase (Austin S, Bingham ET, Matthews DE, Shahan MN, Will J, Burgess RR, Euphytica 85:381-393). Cluster analysis and principal components analysis (PCA) of the Biolog GN metabolic fingerprints indicated consistent differences in substrate utilization between the parental and lignin peroxidase transgenic alfalfa rhizosphere bacterial communities. Cluster analysis of ERIC-PCR fingerprints of the bacterial communities in Biolog GN substrate wells revealed consistent differences in the types of bacteria (substrate-specific populations) enriched from the rhizospheres of each alfalfa genotype. Comparison of ERIC-PCR fingerprints of bacterial strains obtained from substrate wells to substrate community ERIC-PCR fingerprints suggested that a limited number of populations were responsible for substrate oxidation in these wells. Results of this study suggest that transgenic plant genotype may affect rhizosphere microorganisms and that the methodology used in this study may prove a useful approach for the comparison of bacterial communities.

Journal Article↗

PubMed1999

Immobilization of highly concentrated cell suspensions using the laminar jet breakup technique

The controlled breakup of a disturbed jet is a well-known technique for monodisperse particle production and has been well-investigated for Newtonian and viscoelastic fluids. For the immobilization of cells in beads of a hydrogel it has been observed that there is a maximum cell concentration beyond which a monodisperse bead distribution cannot be reached. Higher concentrations lead to irregular jet breakup and thus to droplet coalescence forming beads with double or triple volume. This maximum cell concentration has been investigated for different operating conditions using latex beads of different diameters as a model substance for cells and baker's yeast as a comparison. The maximum cell concentration where a monodisperse size distribution could still be achieved was increased using an electrostatic droplet dispersion system.

Journal Article↗

PubMed1999

Analysis of nifH gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon cascade mountain range.

Nitrogen-fixing microbial populations in a Douglas fir forest on the western slope of the Oregon Cascade Mountain Range were analyzed. The complexity of the nifH gene pool (nifH is the marker gene which encodes nitrogenase reductase) was assessed by performing nested PCR with bulk DNA extracted from plant litter and soil. The restriction fragment length polymorphisms (RFLPs) of PCR products obtained from litter were reproducibly different than the RFLPs of PCR products obtained from the underlying soil. The characteristic differences were found during the entire sampling period between May and September. RFLP analyses of cloned nifH PCR products also revealed characteristic patterns for each sample type. Among 42 nifH clones obtained from a forest litter library nine different RFLP patterns were found, and among 64 nifH clones obtained from forest soil libraries 13 different patterns were found. Only two of the patterns were found in both the litter and the soil, indicating that there were major differences between the nitrogen-fixing microbial populations. A sequence analysis of clones representing the 20 distinct patterns revealed that 19 of the patterns had a proteobacterial origin. All of the nifH sequences obtained from the Douglas fir forest litter localized in a distinct phylogenetic cluster characterized by the nifH sequences of members of the genera Rhizobium, Sinorhizobium, and Azospirillum. The nifH sequences obtained from soil were found in two additional clusters, one characterized by sequences of members of the genera Bradyrhizobium, Azorhizobium, Herbaspirillum, and Thiobacillus and the other, represented by a single nifH clone, located between the gram-positive bacteria and the cyanobacteria. Our results revealed the distinctness of the nitrogen-fixing microbial populations in litter and soil in a Douglas fir forest; the differences may be related to special requirements for degradation and mineralization processes in the plant litter.

Bacteria↗

PubMed1998

A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples.

Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection of Pseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection of Pseudomonas (sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas 16S rRNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species. The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool for Pseudomonas population structure analyses and taxonomic confirmations.

DNA Primers↗

PubMed1998

Medial meniscal cyst imitating a tumor, with compression of the saphenous nerve.

We report a case of large medial meniscal cyst responsible for symptomatic saphenous nerve compression in a 49-year-old male with a history of mild trauma to the affected knee. We are not aware of any similar cases in the literature. The lesion was delineated by ultrasonography and even more clearly by magnetic resonance imaging. At surgery, the saphenous nerve was seen to be displaced by the cyst. Cystectomy and partial meniscectomy were performed. The outcome was favorable.

Bone Neoplasms↗

PubMed1997

[D-Arg1,D-Trp5,7,9,Leu11]substance P: a novel potent inhibitor of signal transduction and growth in vitro and in vivo in small cell lung cancer cells.

[D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP) was identified out of a panel of novel SP analogues as the most potent inhibitor of small cell lung cancer (SCLC) cell growth. This analogue inhibited proliferation of H-510 and H-69 SCLC cells in liquid culture and in semisolid media (IC50, 5 microM). Colony formation stimulated by multiple neuropeptides, including vasopressin and bradykinin, was also blocked by [D-Arg1,D-Trp5,7,9,Leu11]SP. This new SP analogue inhibited vasopressin- or bradykinin-induced Ca2+ mobilization and mitogen-activated protein kinase activation. Administration of [D-Arg1,D-Trp5,7,9,Leu11]SP inhibited the growth of an H-69 xenograft in nude mice. Our results support the hypothesis that SP analogue broad-spectrum neuropeptide antagonists could be of therapeutic value in SCLC.

Animals↗

PubMed1997

Propionibacterium avidum sacroiliitis and osteomyelitis.

The anaerobic Gram-positive bacterium Propionibacterium avidum is a common inhabitant of the skin with low pathogenicity. We report a case of P. avidum sacroilitis, psoas abscess and osteomyelitis in a 67-year-old male who had recently undergone surgical repair of an inguinal hernia. The organism was recovered from blood cultures, a bone biopsy specimen and specimens from the abscess. The spectrum of bone and joint infections caused by Propionibacterium is discussed. Infection by Propionibacterium spp. should be considered in patients with bone and joint infections.

Aged↗

PubMed1997

Lipid mobilization and gluconeogenesis in plants: do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis.

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD+ regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD+ regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

Gluconeogenesis↗

PubMed1995

Glyoxysomal malate dehydrogenase and malate synthase from soybean cotyledons (Glycine max L.): enzyme association, antibody production and cDNA cloning.

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD+ oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (Mr) of 670,000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M(r) 510,000 and of gMDH as a dimer of M(r) 73,000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X6)HL motif also found in plant and mammalian thiolases.

Amino Acid Sequence↗

PubMed1995

A synthetic tumor necrosis factor-alpha agonist peptide enhances human polymorphonuclear leukocyte-mediated killing of Plasmodium falciparum in vitro and suppresses Plasmodium chabaudi infection in mice.

A peptide corresponding to residues 70-80 of the TNF-alpha polypeptide was synthesized and shown to enhance human PMN-mediated killing of Plasmodium falciparum in vitro and reduced the Plasmodium chabaudi parasitemia in mice. Studies of the mechanism of action showed that the peptide, TNF(70-80), stimulated and primed PMN for an increased respiratory burst and release of granule constituents in response to a second agonist. The PMN-stimulatory activity of the peptide was inhibited by mAbs against the p55 and p75 TNF receptors and a TNF-neutralizing mAb. Analysis of PMN receptor expression showed that CR3 (CD18/CD11b) and Fc gamma RIII were upregulated by TNF(70-80), which was consistent with the peptide's ability to enhance parasite killing by PMN. The peptide, unlike TNF, did not increase the expression of adhesion molecules on endothelial cells and failed to promote binding of P. falciparum-infected erythrocytes to endothelial cells. TNF(70-80) also inhibited the TNF-induced increase in adhesion of P. falciparum-infected erythrocytes to endothelial cells. The results demonstrate that the host-protective effects of TNF can be retained while toxic effects are eliminated using a selected, characterized subunit of the cytokine.

Animals↗